Echinacea is a herb well known for its immune stimulatory activity. Echinacea formulations are widely available to the public and are commonly taken for the prevention and treatment of colds and flu.
Traditionally, herbal medicines were available in liquid form as tinctures, teas and the like. However, liquids are not favored by the public for reasons such as taste and convenience. Thus, herbal preparations are generally commercially available in tablet or capsule form. Many tablets are comprised simply of ground dried herb. A disadvantage of ground herbal products is that they contain large amounts of inert plant matter as compared to the active components. Thus relatively large numbers of relatively large tablets need to be consumed for a patient to obtain a therapeutic dose of active ingredients. This may meet with consumer resistance and lead to non-compliance. As such, formulations are generally classified simply by weight of the source herb, the actual content of purported active is not only unknown but can vary significantly from batch to batch.
Other preparations are available in the form of concentrated extracts which are obtained by hydroalcoholic extraction of the herb. An advantage of the concentrated extract is that the soluble constituents of the herb, including the actives, are separated from the inert plant material, thereby decreasing the total volume of material which must be ingested to obtain a therapeutic dose. Concentrated extracts are often standardized to a concentration of a marker compound. Herbal extracts are ideally standardized with respect to the level of an active ingredient such that the effects of batch to batch variations may at least partially be negated, whilst providing an indication as to potency of a particular dosage unit. Difficulties arise however, where the identity of the active compounds are unknown or uncertain. Many manufacturers of Echinacea formulations produce extracts standardized to echinacoside or cichoric acid.
There are many hundreds of commercial preparations identified by the term Echinacea, the content and chemical profile of which preparations vary significantly. There are a number of reasons for such variance. First, there are three species of Echinacea, which are used commercially; E. purpurea, E. angustifolia and E. paffida. Different species contain different levels of active agents and commercial preparations may be sourced from any one or more of these species. Further, different parts of the plant contain different levels of active agents and various parts of the plant such as the root or the aerial parts (leaf, flower or stem) may be and are used as a source of Echinacea preparations. Commercial preparations are commonly sourced from the juice obtained from the aerial parts of E. purpurea. E. purpurea has been favoured due to its ease of cultivation and lower cost. Other preparations are sourced from fresh or dried whole plant, aerial parts or root of one or more of the above three species, hydroalcoholic extracts, or mixtures of any of these.
The literature is replete with reports of studies which have been conducted in an effort to learn more about the action of Echinacea and also to test the efficacy of various preparations. Despite the amount of available literature on the subject, it is still not clear from the available evidence as to which constituents or combinations thereof are responsible for the pharmacological activity of Echinacea. Many in vivo studies are in conflict and inconclusive. Some trials concluded that Echinacea was effective in treating the common cold, whereas other studies could draw no such conclusion. In many cases, trials were conducted on samples which were poorly, if at all, quantified with respect to potentially active components. Thus it is difficult to draw any meaningful conclusions from such trials. In other cases conclusions as to in vivo activity have been drawn from in vitro studies without due consideration being given to the inherent difficulties associated with such extrapolation.
The constituents of Echinacea which have received the most attention are the caffeic acid derivatives such as echinacoside and cichoric acid, polysaccharides and alkylamides (also called alkamides). Echinacoside has been reported to have antibacterial and antiviral activity. However, there is no evidence to date that supports any immunomodulatory activity. On the other hand, cichoric acid has been observed to cause a marked stimulation of phagocytosis in vitro and in vivo. This suggests cichoric acid may be an important contributor to the therapeutic effect of Echinacea. However, the present inventors have observed that cichoric acid and echinacoside exhibit very poor passage across Caco-2 cell monolayers. Such monolayers are used to model the absorption of compounds across the intestinal barrier. The present inventors have therefore suggested that caffeic acid conjugates such as cichoric acid and echinacoside, due to poor permeability across the is intestinal barrier, may not be responsible for the observed in vivo effects of Echinacea. 
In vitro research has also suggested that the polysaccharides promote T cell activity and many commercial Echinacea products are promoted on this basis. However, the relationship between this in vitro work and the therapeutic effects of Echinacea have been questioned for a number of reasons. For example, research has shown that absorption in the gut of similar polysaccharides found in Aloe Vera juice is about 1%. This suggests that insufficient levels of polysaccharides from Echinacea would be absorbed to have any biological effect. Further as polysaccharides are sparingly soluble in ethanol, it is believed that negligible quantities of polysaccharides would be present in hydroalcoholic extracts. However, in vivo studies have shown that such hydroalcoholic extracts do indeed exhibit an immunomodulating effect.
The alkylamides are found in E. angustifolia and E. purpurea but are largely absent from E. pallida. These alkylamides are unsaturated butylamides and may be classified into two groups, the 2-ene and 2,4-diene alkylamides. These alkylamides may be represented by the following formulae:
wherein R1 is H or Me; R2 is a C6 to C10 saturated or unsaturated carbon chain and R3 is a C5 to C9 saturated or unsaturated carbon chain.
The ratio of these two classes of alkylamides may vary considerably depending upon the source. Both root and aerial parts of E. purpurea contain low levels of the 2-enes whereas E. angustifolia root contains a relatively large proportion of the 2-enes.
In view of the significance of Echinacea as both a commercial and therapeutic product, many manufacturers of herbal medicines have sought to develop products which they can claim to have superior efficacy or potency over a competitor's product. Some manufactures have sought to formulate synergistic combinations of Echinacea with other extracts such as Panax Ginseng, Goldenseal or Cat's Claw. Other preparations combine an Echinacea extract with vitamins and minerals which are believed to assist in is the treatment of colds and flu. Combining an Echinacea extract with agents known to increase the biological effectiveness of biologically active agents has also been proposed. Such agents include the salts of succinic acid.
Another approach with a view to improving commercially available products has been to provide different methods of formulation. As mentioned above herbal medicines were traditionally available as liquids. It is generally believed that a liquid system is a more efficient delivery system than a solid system for reasons of dissolution and solubility. However, as also mentioned earlier, there is consumer resistance to such delivery methods. Thus there have been attempts to formulate an Echinacea product in capsule form which may have improved solubility and absorption properties. To this end microencapsulated Echinacea formulations have appeared in the market place. Microencapsulation is well known in the pharmaceutical arts to increase the solubility of an active compound and involves encapsulating droplets of liquids by a thin coating.
In the light of the current information and understanding of those in the art regarding Echinacea, the present inventors have undertaken studies to investigate those compounds in Echinacea which not only exhibit an immunomodulatory effect but are also bioavailable. For example, one such study investigated the effect of Echinacea intake in human subjects on the immune response through altered expression of heat shock protein (hsp70) in leucocytes, increased white blood cell counts and improved erythrocyte antioxidant defenses. The Echinacea was a tablet form of a ethanolic extract of the root of E. purpurea and E. angustifolia. The results after a two week dosing regime showed that Echinacea affected the immune system by increasing white cell counts and the response of hsp70 in leucocytes to heat shock as well as decreasing erythrocyte haemolysis. The inventors also identified the presence of an alkylamide in the blood of all subjects, strongly suggesting that the alkylamide was responsible for the observed effects. The presence of the alkylamide was inconsistent with earlier observations that the alkylamides in solid E. purpurea preparations are rapidly metabolized in the liver and are poorly bioavailable.
In another study in which the present inventors participated, the is modulation of macrophage immune response by an ethanolic extract of E. purpurea and E. angustifolia roots was investigated.
Macrophages play an essential role in the development of specific and non-specific immune responses. Upon activation by a variety of stimuli macrophages release a number of products having biological activity. Such products include cytokines, such as TNF-α, NF-κB and nitric oxide (NO). Macrophages undergo a process of cellular “activation” which is associated with morphological, functional, and biochemical changes in the cells in response to inflammatory signals or antigens. One prominent characteristic of activated macrophages is their increased capacity to release pro-inflammatory and cytotoxic mediators, which help aid in the resolution of infection or inflammation. As a prelude to macrophage activation LPS must bind to a receptor on the macrophage cell surface. Several LPS receptors have been identified in different macrophages with the most thoroughly studied receptor being the CD14 receptor. The mouse peritoneal macrophage cell line, RAW 264.7 used in this study possesses the CD14 receptor.
In this study, the activity of an ethanolic Echinacea extract containing both caffeic acid conjugates and alkylamides on NF-κB, TNF-α and NO were compared to that of the alkylamide fraction alone, cichoric acid and a synthetic 2-ene alkylamide and a synthetic 2,4-diene alkylamide. The synthetic alkylamides correspond to the major 2-ene and 2,4-diene alkylamides found in Echinacea. These alkylamides are illustrated in FIG. 1 as compounds 6 and 3 respectively.
The results showed that all components tested except the 2-ene alkylamide significantly decreased lipopolysaccharide (LPS) stimulated NF-κB levels. Only cichoric, the Echinacea extract and the alkylamide mixture significantly decreased TNF-α production under LPS stimulated conditions in macrophages. Only the alkylamide mixture decreased LPS stimulated NO production. The mixture of alkylamides in the Echinacea ethanolic liquid extract did not respond in the same manner in the assays as the individual alkylamides investigated.
These results demonstrate that the alkylamides, cichoric acid and Echinacea are an effective modulator of macrophage immune responses in vitro. However, it is believed that it is unlikely that cichoric acid would have any observable effect in vivo in view of it's low permeability across Caco-2 monolayers.
The present invention relates to the surprising and unexpected discovery by the inventors that the 2-ene alkylamides are metabolized by the liver at much lower rates that the 2,4 diene alkylamides and still further that the 2-ene alkylamides can actually inhibit 2,4-diene liver metabolism.